EP0446315A1 - Produit recombinant. - Google Patents

Produit recombinant.

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Publication number
EP0446315A1
EP0446315A1 EP90912919A EP90912919A EP0446315A1 EP 0446315 A1 EP0446315 A1 EP 0446315A1 EP 90912919 A EP90912919 A EP 90912919A EP 90912919 A EP90912919 A EP 90912919A EP 0446315 A1 EP0446315 A1 EP 0446315A1
Authority
EP
European Patent Office
Prior art keywords
pai
molecule
expression product
glycosylated
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP90912919A
Other languages
German (de)
English (en)
Other versions
EP0446315A4 (en
EP0446315B1 (fr
Inventor
Peter Lawrence Whitfeld
Michael Andrew Richardson
Clive Leighton Bunn
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Biotech Australia Pty Ltd
Inhibin Pty Ltd
Original Assignee
Biotech Australia Pty Ltd
Biotechnology Australia Pty Ltd
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Filing date
Publication date
Application filed by Biotech Australia Pty Ltd, Biotechnology Australia Pty Ltd filed Critical Biotech Australia Pty Ltd
Publication of EP0446315A1 publication Critical patent/EP0446315A1/fr
Publication of EP0446315A4 publication Critical patent/EP0446315A4/en
Application granted granted Critical
Publication of EP0446315B1 publication Critical patent/EP0446315B1/fr
Anticipated expiration legal-status Critical
Expired - Lifetime legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8132—Plasminogen activator inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/036—Fusion polypeptide containing a localisation/targetting motif targeting to the medium outside of the cell, e.g. type III secretion
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/90—Fusion polypeptide containing a motif for post-translational modification
    • C07K2319/91—Fusion polypeptide containing a motif for post-translational modification containing a motif for glycosylation

Definitions

  • the invention relates to enhanced secretion of PAI-2, to recombinant polynucleotide constructs suitable for providing enhanced secretion of PAI-2, to the expression products of those constructs and to their uses, as well as a 414 amino acid form of PAI-2, a synthetic signal peptide used in the preparation of glycosylated, secreted PAI-2 and 60 kD glycosylated, secreted, recombinant PAI-2.
  • PAI-2 Plasminogen activator (PA) inhibitor Type 2 (PAI-2) is one of four immunologically distinct groups of PA inhibitors. PA inhibitors are members of the serpin gene family.
  • PAI-2 also termed minactivin, has been purified from the human monocytic cell line U937 (International Patent Application PCT/AU85/00191 published as WO86/01212 and PCT/AU87/00068 published as WO87/05628) . It has also been detected in pregnancy plasma and in the conditioned medium of several human cells including peripheral blood monocytes, HT1080 fibrosarcoma cells and HEp3 laryngeal carcinoma cells, Distinct molecular weight species of PAI-2 have been identified:
  • a 46kD form is non-g]ycosylated and primarily cell associated. This form has been found in lysates of U937 cells and purified from the conditioned medium of phorbol ester (PMA) stimulated U937 cells (International Patent Application No. PCT/AU87/00068) . - 2 - A 60kD glycosylated form has been found to be secreted by U937 cells (Genton et al. , 1987) as well as being present in the maternal plasma of pregnant women (Lecander and Astedt, 1986). The non-glycosylated 46kD intracellular form accounts for 80-90% of PAI-2 synthesized in U937 (Genton et al. , 1987) and HT1080 cells (Medcalf et al.. 1988)
  • PAI-2 is produced in very small amounts in vivo and as such is difficult to purify and characterise by conventional biochemical approaches.
  • the recent cloning of the gene for PAI-2 and its expression in bacterial cells now allows the production of significant quantities of purifed 46kD PAI-2 which is needed to evaluate its biological efficacy in clinical applications.
  • this bacterial material is not glycosylated, nor modified post-translationally in a manner analogous to that secreted by human cells. Therefore it is desirable to produce glycosylated forms of PAI-2 using transfected mammalian cells, since the two forms of PAI-2 may differ in their biological activities e.g.
  • PAI-2 binding affinity for urokinase, PA specificity, immunogenicity, in vivo half-life etc.
  • the native PAI-2 gene has previously been expressed in a number of heterolo ⁇ ous mammalian expression systems (International Patent Application No. PCT/AU87/00068) . Although PAI-2 is synthesized in these systems, expression levels are low, and the majority of the product (approx. 90%) is non-glycosylated and intracellular. PAI-2 produced in this form is a suitable molecule for prophylactic, therapeutic and/or diagnostic uses but ; ts use is limited by the quantities obtainable and limited glycosylation.
  • heterologous hydrophobic signal domain to the NH -terminus of a cytoplasmic protein may result in certain circumstances in the translocation of the cytoplasmic protein across the endoplasmic reticulum (Hiebert and Lamb, 1988) .
  • Translocation across the ER is the first step along the secretory pathway.
  • the nature of the heterologous signal peptide e.g. whether or not it is cleaved from the protein
  • the inherent properties of the cytoplasmic protein e.g. the presence of other transport or membrane retention signals
  • Transient NH 2 -terminal signal sequences found on most secreted proteins are identified by three distinct regions - a basic N-terminal region which may contain charged residues, a central hydrophobic core and a C-terminal region containing the signal peptidase recognition site.
  • PAI-2 not only facilitates secretion of the glycosylated 60 kD form of PAI-2 but also directs correct processing of the signal.
  • PAI-2 has been secreted either with or without its N-terminal methionine residue.
  • PAI-2 variant and “variant of PAI-2” refer to a molecule derived from PAI-2 by alteration to the NH spirit-terminus thereof to provide an efficient signal sequence for secretion of the glycosylated molecule from a host cell.
  • parenteral as used herein includes subcutaneous injections, intravenous, or intramuscular injection, or infusion techniques.
  • synthetic signal peptide refers to a peptide which is made in vitro or in vivo by translating a synthesized oligo-deoxyribonucleotide sequence.
  • a process for the preparation of recombinant, glycosylated, secreted PAI-2 or recombinant glycosylated secreted PAI-2 without its N-terminal methionine comprises: providing a construct comprising a first polynucleotide molecule encoding PAI-2 or PAI-2 without its N-terminal methionine; and attaching a polynucleotide molecule encoding a transient signal sequence to the 5' end of the first polynucleotide molecule, such that the resulting hybrid protein expressed from the construct will consist of a transient signal sequence attached to the NH_-terminal of PAI-2 or of PAI-2 without its N-terminal methionine; and expressing the construct in a eukaryotic host cell.
  • the signal peptide may be attached to the N-terminal methionine of PAI-2 or alternatively the N-terminal methionine may be deleted, and the signal peptide attached to the next residue, viz glutamic acid.
  • the signal peptide is designed to be cleaved during translocation of the PAI-2 molecule across the endoplasmic reticulum of the host cell.
  • the signal peptide may be a synthetic signal peptide.
  • the synthetic signal sequence is designed so that it has three distinct regions - a basic N-terminal region which may contain charged residues, a central hydrophobic core and a C-terminal region containing the signal peptidase recognition site. Attempts at designing signal peptidase cleavage sites can be assisted using the method of von Heijne (1986).
  • a preferred synthetic peptide is:
  • the signal peptide may be naturally occurring.
  • a preferred natural signal peptide is the signal peptide from the protein human ⁇ -1-antitrypsin:
  • a recombinant glycosylated PAI-2 encoding construct comprising a polynucleotide molecule encoding a transient signal peptide attached to the NH 2 -terminal methionine or the NH_-terminal glutamic acid of PAI-2.
  • the construct is a DNA molecule.
  • a variant of PAI-2 comprising an altered NH 2 -terminus wherein the NH 2 -terminus of PAI-2 is altered to provide an efficient signal peptide.
  • the alteration is in the first 22 amino acids of the amino acid sequence of PAI-2.
  • amino acids are replaced by a more hydrophobic amino acid selected from Leu, Phe, Ala, Met, Thr, Ser, lie and Val.
  • a fourth embodiment of this invention there is provided a polynucleotide molecule encoding a PAI-2 variant of the third embodiment.
  • the polynucleotide molecule is a DNA molecule.
  • the signal encoding sequences are prepared by DNA synthesis regardless of whether they are synthetic or naturally occurring signal sequences.
  • a recombinant DNA molecule comprising a polynucleotide molecule of the second embodiment or of the f-mrth embodiment which polynucleotide molecule is a DNA molecule, and vector DNA.
  • the vector DNA is plasmid DNA.
  • Preferred plasmid vectors of the invention include pGEM4Z, pSVL, pBTA613, pBTA830 and baculovirus transfer vectors such as pAC373.
  • Preferred recombinant DNA molecules of the invention include pBTA822, pBTA823, pBTA825, pBTA826, pBTA827, pBTA828 and pBTA839.
  • a transformed host cell transformed by a recombinant DNA molecule of the sixth embodiment.
  • Typical host cell lines are derived from eukaryotic organisms and include monkey kidney COS cells, the monkey kidney cell line Vero, Chinese Hamster Ovary (CHO) cells, the human histiocytic lymphoma U937 cell line, derivatives of CHO-K1 cells, for example DG44, the hamster kidney cell line BHK-21, the human kidney cell line 293, the human epithelial cell derived line HeLa S3 and the human monocyte cell line HL60 and cell lines derived from the insects Spodoptera fruoiperda and Bombyx mori.
  • monkey kidney COS cells the monkey kidney cell line Vero
  • Chinese Hamster Ovary (CHO) cells the human histiocytic lymphoma U937 cell line
  • derivatives of CHO-K1 cells for example DG44
  • the hamster kidney cell line BHK-21 the human kidney cell line 293, the human epithelial cell derived line HeLa S3 and the human monocyte cell line HL60 and cell lines derived
  • the transformation is carried out by the calcium phosphate method or by electroporation.
  • an expression product of a transformed host cell of the seventh embodiment is glycosylated PAI-2 or a variant of PAI-2.
  • the invention also provides the 414 amino acid form of PAI-2 which lacks the N-terminal methionine of native
  • PAI-2 in glycosylated and unglycosylated form.
  • the invention provides for the first time recombinant, glycosylated, secreted 60 kD PAI-2.
  • composition comprising an effective amount of an expression product of the eighth embodiment, or of the 60 kD glycosylated, secreted recombinant PAI-2, or of the 414 amino acid form of PAI-2 together with a pharmaceutically acceptable carrier, diluent, excipient, and/or adjuvant.
  • a process for preparing a recombinant DNA molecule of the sixth embodiment which process comprises inserting a DNA molecule of the second embodiment or of the fourth embodiment into vector DNA.
  • a process for preparing a transformed host cell of the seventh embodiment which process comprises making a host cell competent for transformation and transforming said competent host cell with a recombinant DNA molecule of the sixth embodiment.
  • a process for preparing an expression product of the eighth embodiment which process comprises culturing a transformed host cell of the seventh embodiment and separating the expression product from the culture.
  • a process for preparing a composition of the ninth embodiment which process comprises admixing an expression product of the eighth embodiment or 60 kD recombinant glycosylated secreted PAI-2, or the 414 amino acid form of PAI-2 with a pharmaceutically acceptable carrier, diluent, excipient, and/or adjuvant.
  • a fourteenth embodiment of this invention there is provided an antibody raised against an expression product of the eighth embodiment or against recombinant secreted glycosylated 60 kD PAI-2, or against the 414 amino acid form of PAI-2, or against a composition of the ninth embodiment.
  • an antibody composition comprising an effective amount of an antibody of the fourteenth embodiment together with a pharmaceutically acceptable carrier, diluent, excipient, and/or adjuvant.
  • a method of passively vaccinating a host in need of such treatment comprises administering an effective amount of an antibody of the fourteenth embodiment or an antibody composition of the fifteenth embodiment., to the host.
  • a process for producing an antibody of the fourteenth embodiment which process comprises immunising an immunoresponsive host with an expression product of the eighth embodiment, or recombinant glycosylated secreted 60 kD PAI-2, or the 414 amino acid form of PAI-2, or a composition of the ninth embodiment.
  • an expression product of the eighth embodiment produced by the process of the twelfth embodiment.
  • an antibody of the fourteenth embodiment produced by the process of the seventeenth embodiment.
  • the antibodies produced may be monoclonal or polyclonal antibodies.
  • the PAI-2 molecules of the invention are used as immunogens according to standard techniques for the preparation of monoclonal antibodies.
  • a diagnostic kit containing an expression product, and/or composition, and/or antibody and/or antibody composition, respectively of the eighth, ninth, fourteenth and fifteenth embodiments, and/or the 414 amino acid form of PAI-2 and/or 60 kD glycosylated secreted recombinant PAI-2, together with positive and negative standards as controls.
  • a reagent for locating and defining the boundaries of tumours in histological specimens or in vivo which reagent compr: es a suitably labelled expression product of the eighth embodiment, or suitably labelled 60 kD recombinant secreted glycosylated PAI-2, or suitably labelled 414 amino acid form of PAI-2.
  • a method of locating and defining the boundaries of tumours in histological specimens or in vivo which method comprises applying or administering a reagent according to the twenty-first embodiment to the specimen or patient respectively and subsequently imaging to determine the site of the concentration of the label.
  • a method of inhibiting tumour invasion or treating tumours comprising administering to a patient requiring such treatment a therapeutically effective amount of an expression product of the eighth embodiment or a composition of the ninth embodiment or 60 kD glycosylated secreted recombinant PAI-2 or the 414 amino acid form of PAI-2 either labelled or unlabelled.
  • a method of treatment of chronic inflammation such as rheumatoid arthritis comprising administering to a patient requiring such treatment a therapeutically effective amount of an expression product of the eighth, embodiment or a composition of the ninth embodiment or recombinant glycosylated secreted 60 kD PAI-2 or the 414 amino acid form of PAI-2 either labelled or unlabelled.
  • Labels to be used in conjunction with 60 kD PAI-2, expression products and 414 amino acid forms of PAI-2 in accordance with this invention are those standardly used in the art for labelling for the purposes of diagnosis, n vitro detection, imaging, or therapy.
  • a method of monitoring chronic inflammation comprising the detection of PAI-2 in samples of body fluids and tissues using antibodies of the fourteenth embodiment or an antibody composition of the fifteenth embodiment.
  • a method of monitoring chronic inflammation comprising using an expression product of the eighth embodiment, recombinant glycosylated secreted 60 kD PAI-2 or the 414 amino acid form of PAI-2.
  • PAI-2 variants expression products 414 amino acid form of PAI-2 and recombinant glycosylated secreted 60 kD PAI-2 produced in accordance with this invention are of use in the following areas: therapy, prophylaxis and diagnosis of inflammatory disease; therapy, prophylaxis and diagnosis of cancer metastasis and proliferation; therapy of transplant and placenta rejection crises and graft-versus-host reactions; therapy and diagnosis of autoimmune diseases and diseases associated with excessive oxygen radical production; therapy and prophylaxis of wound and bone healing disorders, tissue damage during or after acute reperfusion, and subarahnoidal bleeding disorders; as topical medicaments for promoting fibrin adhesion, promoting healing of wounds and burns; treating asthma and treating or preventing diseases associated with high leukocyte activity; and suppression of the monocyte-macrophage system.
  • the amount of expression product, 60 kD glycosylated secreted recombinant PAI-2 or 414 amino acid form of PAI-2 that may be combined with carrier to produce a single dosage form will vary depending upon the condition being treated, the host to be treated and the particular mode of administration. It will be understood, also, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific protein product or antibody employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, the particular state being treated and the severity of the particular condition undergoing treatment.
  • compositions of the present invention may be administered parenterally or topically or potentially via mucosal routes in dosage unit formulations containing conventional, non-toxic, pharmaceutically acceptable carriers, diluents, adjuvants and/or excipients as desired.
  • injectable preparations for example, sterile injectable aqueous or oleagenous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, a solution in 1,3-butanediol.
  • acceptable vehicles and solvents that may be employed are water.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including sythetic mono- or diglycerides,
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • pharmaceutically acceptable adjuvant can mean either the standard compositions which are suitable for human administration or the typical adjuvants employed in animal vaccinations.
  • Suitable adjuvants for the vaccination of animals include but are not limited to oil emulsions such as Freund's complete or incomplete adjuvant (not suitable for livestock use), Marcol 52: Montanide 888 (Marcol is a Trademark of Esso. Montanide is a Trademark of SEPPIC,
  • Adjuvant 65 containing peanut oil, mannide monooleate and aluminium monostearate
  • mineral gels such as aluminium hydroxide, aluminium phosphate, calcium phosphate and alum
  • surfactants such as hexadecylamine, octadecylamine, lysolecithin, dimethyldioctadecylammonium bromide,
  • the expression products of the present invention can also be administered following incorporation into liposomes or other micro-carriers, or after conjugation to polysaccharides, proteins or polymers or in combination with Quil-A to form "Iscoms" (Immunostimulating complexes) (Morein ei_al. , Nature I , 457-460 [1984]).
  • Other adjuvants suitable for use in the present invention include conjugates comprising the expression _ roduct together with an integral membrane protein of prokaryotic origin, such as TraT. (See PCT/AU87/00107)
  • compositions for topical administration include creams, ointments and pastes.
  • the ingredients that constitute the base of ointments e.g. petrolatum, waxes
  • ointments e.g. petrolatum, waxes
  • powdered drug components are added and the mass stirred with cooling.
  • the product is then passed through a roller mill to achieve the - 14 - particle-size range desired for the dispersed solid.
  • Pastes are ointments with relatively large, dispersed solid content, and are prepared similarly.
  • Creams are semisolid emulsions, either water-in-oil or oil-in-water.
  • a solid ingredient can be added to the appropriate phase before emulsification or may be dispersed at some point after the emulsification step.
  • Topical dosage forms include disc dosage form systems that have been used for transdermal delivery of therapeutic agents. They provide uniform and prolonged drug release.
  • compositions of the invention either orally, rectally or possibly even vaginally.
  • Compositions which could be used in these ways could be prepared as follows:
  • Suppositories for rectal or vaginal administration of the compositions of the invention can be prepared by mixing the composition with a suitable nonirritating exipient such as cocoa butter, theobroma oil, glycerinated gelatin or polyethylene glycols which are solid at ordinary temperatures but liquid at rectal or vaginal temperature or by contact with fluids present in the appropriate cavity and will therefore melt in the rectum or vagina and release the drug.
  • a suitable nonirritating exipient such as cocoa butter, theobroma oil, glycerinated gelatin or polyethylene glycols which are solid at ordinary temperatures but liquid at rectal or vaginal temperature or by contact with fluids present in the appropriate cavity and will therefore melt in the rectum or vagina and release the drug.
  • Solid dosage forms for oral administration may include capsules, tablets, pills, powders, and granules.
  • expression products, recombinant glycosylated secreted 60 kD PAI-2 or 414 amino acid form of PAI-2 may be admixed with at least one inert diluent such as sucrose, lactose starch or hydrolysed gelatin.
  • inert diluent such as sucrose, lactose starch or hydrolysed gelatin.
  • Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate.
  • the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.
  • Liquid dosage forms for oral administration may include nanoparticles, microcapsules, LTB conjugates, cholera or its B subunit as a conjugate, or vitamin B12 conjugates in pharmaceutically acceptable emulsions, syrups, solutions, suspensions, and elixirs containing inert diluents commonly used in the art, such as water.
  • compositions may also comprise adjuvants, such as wetting agents, emulsifying and suspending agents or TraT as a conjugate, and sweetening, flavouring, and perfuming agents including sugars such as sucrose, sorbitol, fructose etc, glycols such as polyethylene glycol, propylene glycol etc, oils such as sesame oil, olive oil, soybean oil etc, antiseptics such as alkylparahydroxybenzoate etc, and flavours such as strawberry flavour, peppermint etc.
  • adjuvants such as wetting agents, emulsifying and suspending agents or TraT as a conjugate
  • sweetening, flavouring, and perfuming agents including sugars such as sucrose, sorbitol, fructose etc, glycols such as polyethylene glycol, propylene glycol etc, oils such as sesame oil, olive oil, soybean oil etc, antiseptics such as alkylparahydroxybenzoate etc, and flavours such as strawberry flavour, peppermint etc.
  • the recombinant constructs of the invention provide expression of secreted and glycosylated forms of PAI-2.
  • Figure 1 shows the sequence of oligonucleotides and encoded signal peptides employed in construction of the in vitro and in vivo PAI-2 expression vectors.
  • Figure 2 shows construction of in vitro expression vectors:
  • FIG. 1 shows construction of mammalian cell expression vectors:
  • Figure 4 shows the products of the PAI-2 gene and signal variants translated in a cell-free system and electrophoresed through an SDS-PAG.
  • Figure 5 shows expression of PAI-2 gene and signal variants in transfected mammalian cells. Aliquots of the medium were immunoprecipitated with an anti-PAI-2 antibody and precipitates run on an SDS-PAG.
  • Figure 6 shows urokinase binding to PAI-2 secreted from transfected COS cells immunoprecipitated with anti-PAI-2 antibodies and electrophoresed through an SDS-PAG.
  • Figure 7 shows DNA and amino acid sequence of preA-PAI2 VIZ PAI-2 plus synthetic signal. Amino acids in capitals indicate the signal sequence.
  • Figure 8 shows DNA and amino acid sequence of preB-PAI-2 VIZ PAI-2 plus ⁇ -1-antitrypsin signal. Amino acids in capitals indicate the signal sequence.
  • Figure 9 shows the digestion of the 60,000 Mr PAI-2 secreted from transfected COS cells with various glycosidases. The products were separated by SDS-PAGE.
  • Figure 10 shows the peaks of radioactivity released during repeated cycles of Edman degradation on labelled 60,000 Mr PAI-2 secreted from COS cells transfected with: A: pBTA 825 (contains PAI-2 gene with synthetic signal) B: pBTA 826 (contains PAI-2 gene with alpha-1 antitrypsin signal)
  • Figure 11 shows the secretion of 60,000 Mr PAI-2 into the medium from a CHO-K1 cell line stably transfected with pBTA 827. The PAI-2 was immunoprecipitated from the medium and then fractionated by SDS-PAGE.
  • the recombinant DNA molecules and transformed host cells of the invention are prepared using standard techniques of molecular biology.
  • Expression products of the invention are obtained by culturing the transformed host cells of the invention under standard conditions as appropriate to the particular host cell and separating the expression product from the culture by standard techniques.
  • the expression product may be used in impure form or may be purified by standard techniques as appropriate to the expression product being produced.
  • compositions of the invention are prepared by mixing, preferably homogeneously mixing, expression product, 60 kD recombinant secreted glycosylated PAI-2 or the 414 amino acid form of PAI-2 with a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant using standard methods of pharmaceutical preparation.
  • the amount of expression product, recombinant glycosylated secreted 60 kD PAI-2, or the 414 amino acid form of PAI-2 required to produce a single dosage form will vary depending upon the condition to be treated, patient to be treated and the particular mode of administration.
  • the specific dose level r any particular patient will depend upon a variety of factors including the activity of the expression product or PAI-2 molecule employed, the agt , body weight, general health, sex, and diet of the patient, time of administration, route of administration, rate of excretion, drug combination and the severity of the condition undergoing treatment.
  • compositions may be administered parenterally or topically, or possibly by inhalation spray, vaginally, orally or rectally in unit dosage formulations containing conventional, non-toxic, pharmaceutically acceptable carriers, diluents, excipients and/or adjuvants as desired.
  • Antibodies are raised using standard vaccination regimes in appropriate hosts. The host is vaccinated with an expression product, recombi n ant glycosylated secreted 60 kD PAI-2 or the 414 amino acic form of PAI-2 or a composition of the invention.
  • the antibody composition is prepared by mixing, preferably homogeneously mixing, antibody with a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant using standard methods of pharmaceutical preparation.
  • the amount of antibody required to produce a single dosage form will vary depending upon the condition to be treated, patient to be treated and the particular mode of administration.
  • the specific dose level for any particular patient will depend upon a variety of factors including the activity of the antibody employed, the age, body weight, general health, sex, and diet of the patient, time of administration, route of administration, rate of excretion, drug combination, and severity of the condition undergoing treatment.
  • the antibody composition may be administered parenterally in unit dosage formulations containing conventional, non-toxic, pharmaceutically acceptable carriers, diluents, excipients and/or adjuvants as desired.
  • Diagnostic kits are prepared by formulating expression product and/or antibodies and/or recombinant glycosylated secreted 60 kD PAI-2 and/or the 414 amino acid form of PAI-2 at appropriate concentration to the substance(s) to be detected with a pharmaceutically acceptable carrier, diluent, excipient and/or adjuvant.
  • a positive control standard of a known concentration of the substance to be detected is prepared similarly.
  • the negative standard comprises carrier, diluent, excipient and/or adjuvant alone.
  • diagnostic kits include a tumour diagnostic wherein the reagent comprises anti-expression product antibodies and the positive control comprises an expression product standard of known concentration.
  • the second signal is the natural signal peptide from the protein human ⁇ -1-antitrypsin:
  • the second variant of PAI-2 was constructed by fusing the human ⁇ -1-antitrypsin signal peptide to PAI-2 without is NH_-terminal methionine. After correct cellular processing a mature PAI-2 molecule of 414 residues having a glutamic acid residue at its NH_-terminus should be generated. Details of the construction of the signal-PAI-2 molecules and their expression in both in vitro and in vivo systems are described below.
  • Oligonucleotides encoding the signa peptides were synthesized on an Applied Biosystems DNA synthesizer (Model 380A) , and purified through a polyacrylamide gel.
  • Complementary oligonuclotide Al and A2 or Bl and B2 or Cl and C2 were mixed in a 1:1 molar ratio and phosphorylated with 5 units T4 polynucleotide kinase in 65mM Tris-Cl pH 7.5, lOmM MgCl 2 , 5mM dithiothreitol, ImM ATP. The mixture was heated to 100°C for 3 minutes and cooled slowly to room temperature t allow annealing to take place.
  • the annealed signal peptide oligonucleotides were ligated to the PAI-2 coding sequences and cloned into the vectors pGEM4Z (Promega) , pSVL (Pharmacia) pBTA613 and pBTA830.
  • the first vector, pGEM4Z is useful for expressin gene products in a cell-free transcription/translation system; pSVL is useful for transient expression in monkey kidney COS cells; pBTA613 and pBTA830 are vectors that can be selected and stably integrated in mammalian cell lines such as Chinese Hamster Ovary (CHO) cells, Vero, BHK and
  • the signal-PAI-2 gene constructs could also be clone into any other vector designed for expression in eukaryotic cells.
  • the band of agarose containing the appropriate fragment was sliced out of the gel, melted at 65°C and the DNA was extracted by passing the diluted material through a NACS column (BRL) as recommended by the supplier. The DNA was then precipitated with ethanol in the presence of carrier tRNA (10 ⁇ g/ml) .
  • Vectors were typically prepared as follows. Plasmid DNA purified by CsCl density gradient centrifugation was digested with the appropriate restriction enzymes, the digest was extracted with an equal volume of phenol/chloroform (1:1) and the DNA precipitated with 2.5 volumes of ethanol. The digested DNA was resuspended in 50mM Tris-Cl pH 9.0, ImM MgCl 2 , O.lmM ZnCl 2 , ImM spermidine and incubated with 1-2 units calf intestinal alkaline phosphatase (Boehringer Mannheim) for 30-60 mins at 37°C. The enzyme was heat killed at 70°C for 15 mins then the DNA was extracted with phenol/chloroform and precipitated with ethanol.
  • Ligations were carried out as follows. Vector and insert DNAs were mixed at a molar ratio of between 1:1 and 1:5 (1:10 if the insert was smaller than lOObp) in ImM ATP, lOmM MgCl 2 , 5rnM DTT, 65mM Tris-Cl pH7.5 in a volume of 10-20 ⁇ l. Ligations were carried out at 16° overnight with 0.5-1 unit T4 DNA ligase (Boehringer Mannheim). Ligation mixes were diluted to 30 ⁇ l with dH_0 and lO ⁇ l removed for transformation into a competent E. coli K12 host (Hanahan, 1985) . Transfor ants were selected by plating onto tryptone-soya agar plates containing lOO ⁇ g/ml ampicillin.
  • Plasmid DNA was extracted from individual colonies (Birnboim and Doly, 1979) and the correct recombinant plasmids identified by restriction analysis.
  • the 5' ends of the PAI-2 gene in pBTA821, 822 and 823 were sequenced to confirm that the signal peptide and methionine start codons were in frame with the remainder of the PAI-2 sequence.
  • Sequencing was carried out on double-stranded plasmid DNA using the Sequenase DNA Sequencing Kit (USB) as described in the instruction manual.
  • the primer used was the T7 primer (Promega) .
  • Plasmid pBTA641 can be derived from pBTA438 as follows. pBTA438 is partially digested with XhoII plus Dral and a 155 fragment isolated and ligated to vector pLK58 cut with Bglll and Smal. The resultant plasmid pBTA446 was linearized with Bglll and ligated to a synthetic double stranded 27 mer oliognucleotide having the sequence ATCT (N) 16 ATGGAG wherein N represents any nucleotide, containing a bacterial ribosome binding site and the initial nucleotides of the native PAI-2 gene, creating plasmid pBTA641.
  • PBTA679 contains a 1.3kb fragment of the PAI-2 gene (including the entire coding region).
  • the 1.3kb fragment was derived from the 1.6kb EcoRI-Dral fragment from pBTA438 by deleting 300b ⁇ of 3' untranslated region between the stop codon and the Dral site (at 1604) using Bal 31 nuclease.
  • the resultant fragment with linkers ligated to either end was subcloned into the Hindlll-BamHI sites of the multiple cloning site derived from pUCl ⁇ (Yanisch-Perron e_ s_l, 1985) .
  • the end-point of the deletion was determined by sequencing.
  • pBTA830 is a mammalian cell expression vector.
  • pBTA830 comprises the following fragments in order: the 345bp Pv ⁇ ll-Hindlll fragment from the SV40 origin (Bethesda Research Laboratories), 51bp Hindlll-EcoRI multiple cloning sites from pUCl ⁇ , 853bp XhoI-BamHI fragment from pMSG (a eukaryotic expression vector from Pharmacia) with EcoRI and Aatll linkers (GAATTC, GGACGTCC, New England Biolabs) attached to either end, 2262bp Aatll-Eagl fragment from pBR327 (Soberon et al.
  • Sau3A fragment from the SV40 small t intron region 293bp Sau3A fragment from the SV40 early polyadenylation region, 288bp Eagl-Sall fragment from pBR327, 345bp PvuII-Hindlll fragment from the SV40 origin, 734bp Hindlll-Bglll fragment encoding mouse dihydrofolate reductase from pSV2-DHFR (ATCC 37146, Subramani et al, 1981), 141bp Sau3A fragment from SV40 small t intron region and 293bp Sau3A from SV40 early polyadenylation region.
  • pBTA613 is a mammalian cell expression vector. Foreign genes are expressed by cloning into the multiple cloning site flanked upstream by the SV40 early promoter and downstream by SV40 polyadenylation signals. pBTA613 comprises the following fragments in order. The 345bp PvuII-Hindlll fragment from the SV40 origin, 51bp Hindlll-EcoRI multiple cloning sites from pUCl ⁇ , 75bp
  • pBTA 821, 622 and 623 the PAI-2 gene is placed downstream from the T7 RNA polymerase promoter.
  • plasmid DNA purified on CsCl density gradients was linearized then transcribed using the commercially available Riboprobe Gene transcription System (Promega) . Where commercially available kits were used experiments were performed according to the manufacturer's instructions.
  • plasmid DNA was linearized by digestion with EcoRI.
  • the EcoRI digested DNA was treated with proteinase K [at 400 ⁇ g/ml] then extracted with phenol/chloroform (1:1), precipitated with 2.5 vols ethanol and resuspended in lOmM Tris-Cl pH ⁇ .0, ImM EDTA.
  • Approximately l ⁇ g linear DNA was transcribed in a 25 ⁇ l reaction containing 1 x transcription buffer (Promega) , lOmM DTT, 0.5mM each ATP, CTP, GTP, UTP, 40 units RNAsin and 7.5 units T7 RNA polymerase (Promega) at 37° for 60 mins.
  • RNA transcripts were subsequently translated in a rabbit reticulocyte lysate cocktail.
  • RNA transcription reaction
  • the reticulocyte lysate translation mix was supplemented with dog pancreatic microsomal vesicles (Promega) .
  • Microsomes are generated from endoplasmic reticulum (ER) and allow signal peptide cleavage, protein translocation and core glycosylation events to be studied. Any core carbohydrates added to asparagine residues in PAI-2 molecules on translocation across the ER are removed by digesting the translation products with endoglycosidase H. The extent of translocation of a protein across the membrane is assessed by incubating the processed in vitro translation products with proteinase K. Any of the protein not fully translocated into the lumen of the microsomal vesicle is liable to be digested by proteinase K.
  • Endoglycosidase H digests were carried out by incubating 2 ⁇ l translation mix plus 4 ⁇ l RIPA buffer (1% Triton X100, 1% sodium deoxycholate, 0.1% SDS, 150mM NaCl, ' 50mM Tris-Cl pH7.5, 5mM EDTA) plus l ⁇ g aprotinin with
  • Proteinase K digests were carried out as follows. 2 ⁇ l translation mix plus 5 ⁇ l phosphate buffered saline plus 0.l ⁇ g/ ⁇ l proteinase K were incubated on ice for 60 mins. In some reactions l ⁇ l 1% Triton X-100 was included. Samples were boiled in 1.5% SDS, 1% 2-mercaptoethanol before electrophoresis on 10% polyacryla ide-SDS gels.
  • Figures 4A and 4B show the results of typical cell-free translation experiments.
  • the autoradiogram in Figure 4B shows that the primary translation product of the native PAI-2 gene (pBTA821) consists of a 43,000 Mr molecule (Fig 4B lane b) . It is not translocated into the microsomes since proteinase K digests the product whether microsomes were present (Fig. 4B lane d) or absent (Fig. 4B lane a). Also the native product is not glycosylated since digestion with endoglycosidase H ( Figure 4A lane d) has no effect on the migration of the Mr 43,000 product. This 43,000 Mr product was also immunoprecipitated with anti-PAI-2 monoclonal antibody.
  • the medium was removed about 42 hrs after glycerol shock and replaced with Eagles minimal essential medium lacking methionine (Flow) supplemented with lOO ⁇ Ci/ml 35S-methionine (Amersham SJ1015 lOOOCi/mmole) . After 4 hrs of metabolic labelling the medium was collected for analysis and the cells washed with an ice cold phosphate buffered saline solution, harvested and lysed. Cells were lysed in 1% Triton X100, 0.1% SDS, 150mM NaCl, 50mM Tris-Cl pH7.5, 5mM EDTA.
  • PAI-2 was immunoprecipitated from aliquots of medium and cell lysates by incubating with 25 ⁇ g/ml anti-PAI-2 mouse monoclonal antibody (MAI-21, Biopool) at 4°C overnight in a solution containing 1% Triton X100 and lO ⁇ g/ml aprotinin.
  • Antibody-PAI-2 complexes were extracted with 20 ⁇ l anti-mouse IgG-agarose beads (Sigma) in lOmM sodium phosphate pH7.2, 0.5M NaCl.
  • the PAI-2 was eluted from the beads with 0.15M NaCl/O.lM glycine pH2.4, analysed by SDS-polyacrylamide gel electrophoresis under reducing conditions and visualized by autoradiography as shown in Figure 5. Some cells were incubated with 5 ⁇ g/ml tunicamycin for 18 hrs prior to and during the labelling, to prevent N-linked glycosylation.
  • Recombinant PAI-2 is seen in Fig 5, lanes f and h as a broad band of Mr 53,000-60,000. These are samples of medium from cells transfected with the synthetic signal-PAI-2 gene (pBTA825) and ⁇ -1-antitrypsin signal-PAI-2 gene (pBTA ⁇ 26) respectively.
  • the medium from mock transfected cells (i.e. no added DNA) and from cells transfected with the vector alone (pSVL) contain no 60,000 Mr material (lanes b and c) . However a small amount of 60,000 Mr material and a larger amount of 43,000 Mr material is present in the medium from pBTA ⁇ 24 transfected cells (lane d) .
  • the 43,000 Mr material appears not to be glycosylated (compare lanes d and e) .
  • the biological activity of the 58-60,000 Mr forms of PAI-2 were determined by a urokinase (uPA) binding assay.
  • Urokinase (45 IU Low Mol Wt, American Diagnostica) was added to an aliquot of medium containing 35S-labelled PAI-2 from transfected COS cells. Binding was allowed to proceed at room temperature for 90 mins. The uPA-PAI-2 complexes were immunoprecipitated from the reaction using goat anti-PAI-2 antibodies or rabbit anti-uPA serum and a solid phase 2nd antibody. Material was eluted from the immunobeads by boiling in a buffer containing 1.5% SDS and 1% (v/v) 2-mercaptoethanol and then analysed under reducing condi ions by SDS-PAGE.
  • FIG. 6 shows the results of such an experiment.
  • the Mr 43,000 nonglycosylated PAI-2 released from COS cells transfected with the native gene (Fig 6, lane c) forms a Mr 76,000 complex when uPA is added (Fig 6, lane d) .
  • the 58-60,000 glycosylated PAI-2 secreted from COS cells transfected with either of the signal-PAI-2 constructs also binds uPA and a complex of Mr around 89,000 is formed (Fig. 6, lanes f and h) .
  • This complex is immunoprecipitable with both anti-PAI-2 and anti-uPA antibodies.
  • the material secreted from COS cells was digested with a variety of endoglycosidases (see Glycanalysis Systems Manual from Genzyme Corp. USA).
  • the 60,000 Mr form was immunoprecipitated from the medium of cells transfected with pBTA826 as described above. Aliquots of this material were digested as follows.
  • 3H-leucine labelled material secreted from transfected COS cells were subjected to immunoprecipitation using the anti-PAI-2 mouse monoclonal antibody (MAI-21, Biopool) as described above.
  • An aliquot of the 35S-Met labelled PAI-2 recovered from the medium was analyzed by SDS-polyacrylamide gel electrophoresis and shown to contain 60,000 Mr material.
  • the H-leu labelled 60,000 Mr forms of PAI-2 were electrophoresed through an SDS-polyacrylamide gel and transferred to Pro Blot PVDF membrane by electroblotting (Matsudaira, 19 ⁇ 7) .
  • a fragment of the membrane containing the 60 kDa PAI-2 was excised and then applied to the Protein sequencer and sequenced as above with modifications to the Sequencer program as described by Speicher, 1989.
  • Cell line CHO-K1 (ATCC CCL 61) is derived from a Chinese hamster ovary cell.
  • Cell line U937 is the human monocyte-derived cell which expresses PAI-2 and from which the PAI-2 gene was isolated (Antalis et al. 1988) .
  • PAI-2 host cells for the expression of PAI-2 include the hamster kidney cell line BHK-21 (ATCC CCL10), the human kidney cell line 293 (ATCC CRL1573), the human epithelial cell-derived line HeLa S3 (ATCC CCL2.2), and the human monocyte cell line HL60 (ATCC CCL 240) and insect cell lines derived from Spodoptera fruoiperda (Sf9, ATCC CRL1711) and Bombyx mori (Maeda et al. 1984).
  • the PAI-2 genes with added signal sequences were inserted into the vector pBTA830 which contains the mouse dhfr gene and the gene conferring resistance to the aminoglycoside antibiotic, G418 ( Figures 3B and 3C) .
  • the resultant plasmids pBTA827, pBTA828, were transfected into the above cell lines by the calcium phosphate method
  • Electroporation was carried out using lO ⁇ g linearized plasmid DNA/107 cells suspended in phosphate buffered saline/272 mM sucrose, pH 7.4, at 4°C, and pulses of 150-300V at a capacitance of
  • transfectants of the CHO-K1 cell line was achieved in the nucleoside-free alpha modification of Eagles medium ( ⁇ MEM) containing 2% (v/v) dialysed foetal bovine serum and 400 ⁇ g/ml G418. This regime selected for both the dhfr and the G418-resistance genes present in the PAI-2 encoding plasmid. Selection of transfectants of other cell lines was done with ⁇ MEM or RPMI 1640 medium (CytoSystems) containing 2-10% foetal bovine serum and 400 ⁇ g/ml G418.
  • the PAI-2 genes were amplified along with cotransfected (and host) dhfr genes by exposure of the cultures to increasing levels of methotrexate from 0.l ⁇ M to 10 ⁇ M over three months as described by Zettlemeisl et .al. (1987).
  • the resultant transfected cultures with stably integrated, amplified PAI-2 genes were examined for the secretion of PAI-2 into serum-free culture medium by an ELISA (Biopool, Sweden) performed according to the manufacturers instructions. Different cultures (pools) containing cells originating from many hundreds of transfection events were assayed by ELISA and the pools with the highest PAI-2 expression were chosen. The identity of the molecular weight forms of PAI-2 secreted from these cultures was verified by immunoprecipitation of culture supernatants and SDS-polyacrylamide gel electrophoresis as described above. High molecular weight, glycosylated forms of PAI-2 are produced by representative cultures as shown in Figure 11.
  • the biological activty of the secreted forms of PAI-2 can be determined usi ⁇ j a coupled photometric assay for plasminogen activator(Cole an and Green, 1981) .
  • the first step of the reaction involves the activation of purified plasminogen to plasmin by uPA.
  • the activity of plasmin is then detected by the formation of a yellow thiophenolate anion.
  • PAI-2 activity is expressed in Ploug units/ml.
  • clones of individual cells from the highest expressing cell pool are isolated by the limiting dilution method (Freshney, 1987) . Cell colonies are assayed for PAI-2 by transferring them to nylon filters as described by Raetz et al. (1982) and detecting PAI-2 by Western blot analysis. Colonies with maximal PAI-2 expression are isolated, re-cloned, and the secreted forms of PAI-2 verified as described above.
  • Cell line DG44 is a derivative of the Chinese hamster ovary cell CHO-K1 and contains a deletion of the gene for dihydrofolate reductase (dhfr. Urlaub et al., 1986). Vero is derived from an African green monkey kidney cell (ATCC CCL81) .
  • the PAI-2 gene with added signal sequence was inserted into the vector pBTA613 which contains the mouse dhfr gene ( Figure 3D) .
  • the resultant plasmid ⁇ BTA839 was transfected into the above cell lines by the calcium phosphate method described, using lO ⁇ g plasmid DNA/2 x 10 cells and a total of 2 x 10 cells, or by electroporation. Electroporation was carried out as described by Barsoum (1990) using the Bio-Rad Gene Pulser.
  • transfectants of cell line DG44 was achieved in the nucleoside-free medium ocMEM (CytoSystems) containing 2% (v/v) dialysed foetal bovine serum and 0.1-0.5 ⁇ M methotrexate.
  • the selection of transfectants of Vero cells was done in the same medium. This procedure selects for transfectants with an elevated number of copies of integrated plasmid.
  • the PAI-2 genes were amplified along with cotransfected (and host) dhfr genes by exposure of the cultures to increasing levels of methotrexate up to 10 ⁇ M as described by Zettlemeisl et al (1987).
  • the resultant transfected culture with stably integrated, amplified PAI-2 genes are examined for the secretion of PAI-2 into the serum-free culture medium by an ELISA for PAI-2 (Biopool, Sweden) performed according to the manufacturers instructions. Single colonies arising from the transfection and pools containing cells originating from many thousands of transfection events are assayed by ELISA and those with the highest PAI-2 expression are chosen. The identity of the molecular weight forms of PAI-2 secreted from these cultures is verified by immunoprecipitation of culture supernatants and SDS-polyacrylamide gel electrophoresis as described above.
  • Clones of individual cells are also isolated from the highest expressing cell pool by limiting dilution method (Freshney, 1987) . Cell colonies are assayed for PAI-2 by transferring them to nylon filters as described by Raetz et al. (1982) and detecting PAI-2 by Western blot analysis. Colonies with maximal PAI-2 expression are isolated, re-cloned, and the secreted forms of PAI-2 verified as described above.
  • PAI-2 does not contain a signal sequence that is efficiently recognized by the protein translocation machinery present in some mammalian cell extracts. This may explain the observations that PAI-2 is predominantly an intracellular protein. The little material that is secreted is often unglycosylated and may be released from the cells by an unknown mechanism which does not involve transport of PAI-2 through the normal secretory pathway of the ER and Golgi. However high Mr forms of PAI-2 have also been observed; hence PAI-2 must have the capacity to translocate into the ER and Golgi (where addition of carbohydrate occurs) . The signal to allow this has not been identified.
  • the secreted PAI-2 is purified using established and published procedures (see International Patent Application No. PCT/AU87/00068) .
  • the expression products, secreted glycosylated recombinant 60 kD PAI-2 and the 414 amino acid form of PAI-2 of the invention are of use as diagnostic or therapeutic substances in the fields of: therapy, prophylaxis and diagnosis of inflammatory disease; therapy, prophylaxis and diagnosis of cancer metastasis and proliferation; therapy of transplant and placenta rejection crises and graft-versus-host reactions; therapy and diagnosis of autoimmune diseases and diseases associated with excessive oxygen radical production; therapy and prophylaxis of wound and bone healing disorders, tissue damage during or after acute reperfusion, and subarahnoidal bleeding disorders; as topical medicaments for promoting fibrin adhesion, promoting healing of wounds and burns; treating asthma and treating or preventing diseases associated with high leukocyte activity; and suppression of the monocyte-macrophage system.
  • M13 phage cloning vectors and host strains nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 3_1 103-119.

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Abstract

L'invention se rapporte à l'inhibiteur d'activateur de plasminogène de type 2 et à son expression comme molécule recombinante dans des lignées de cellules eukaryotiques sous la forme d'une molécule sécrétée glycosylée, aux structures permettant son expression, à des cellules hôtes effectuant son expression, à des compositions le contenant, à des procédés de traitement, de prophylaxie et de diagnostic l'utilisant et à des anticorps dressés contre lui. L'invention se rapporte également à une forme de 414 acides aminés du PAI-2, dans laquelle le résidu de méthionine de terminaison N est supprimé, à une forme recombinante sécrétée glycosylée de 60 kD du PAI-2 et à des compositions et des procédés utilisant ces molécules. L'invention se rapporte en outre à un nouveau peptide de signal synthétique.
EP90912919A 1989-09-05 1990-09-04 Procede de preparation de pai-2 Expired - Lifetime EP0446315B1 (fr)

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AU2299592A (en) * 1991-07-02 1993-02-11 Children's Medical Center Corporation Treatment of periodontal disease with protease inhibitors
WO1995013094A1 (fr) * 1993-11-10 1995-05-18 Bristol-Myers Squibb Company Traitement des affections inflammatoires d'origine bacterienne
US6458574B1 (en) * 1996-09-12 2002-10-01 Transkaryotic Therapies, Inc. Treatment of a α-galactosidase a deficiency
US6083725A (en) * 1996-09-13 2000-07-04 Transkaryotic Therapies, Inc. Tranfected human cells expressing human α-galactosidase A protein
WO1999049887A1 (fr) * 1998-04-01 1999-10-07 Biotech Australia Pty. Limited Utilisation d'inhibiteurs de la protease pour traiter les blessures cutanees
AUPP508798A0 (en) 1998-08-05 1998-08-27 Biotech Australia Pty Limited Method of treating psoriasis
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US7325244B2 (en) * 2001-09-20 2008-01-29 Keen Personal Media, Inc. Displaying a program guide responsive to electronic program guide data and program recording indicators
US20040071686A1 (en) 2002-04-25 2004-04-15 Treco Douglas A. Treatment of alpha-galactosidase A deficiency
US20040248262A1 (en) * 2003-01-22 2004-12-09 Koeberl Dwight D. Constructs for expressing lysomal polypeptides
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US4923807A (en) * 1984-05-18 1990-05-08 New England Medical Center Hospitals Inc. Arg-Serpin human plasminogen activator inhibitor designated PAI-2
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